PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours.

Although adoptive T-cell therapy has shown remarkable clinical efficacy in haematological malignancies, its success in combating solid tumours has been limited. Here, we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T-cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8+ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling, thereby enhancing both CAR T-cell activation and homing to CXCL9/10-expressing tumours to eradicate HER-2+ mammary tumours in vivo. Our findings define PTPN2 as a target for bolstering T-cell-mediated anti-tumour immunity and CAR T-cell therapy against solid tumours.

A predictive classifier of poor prognosis in transplanted patients with juvenile myelomonocytic leukemia: a study on behalf of the Société Francophone de Greffe de Moelle et de Thérapie Cellulaire.

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm requiring hematopoietic stem cell transplantation (HSCT) in most cases. We retrospectively analyzed 119 JMML patients who underwent first allogeneic HSCT between 2002 and 2021. The majority (97%) carried a RAS-pathway mutation, and 62% exhibited karyotypic alterations or additional mutations in SETBP1, ASXL1, JAK3 and/or the RAS pathway. Relapse was the primary cause of death, with a 5-year cumulative incidence of 24.6% (95%CI: 17.1-32.9). Toxic deaths occurred in 12 patients, resulting in treatmentrelated mortality (TRM) of 9.0% (95%CI: 4.6-15.3). The 5-year overall (OS) and event-free survival were 73.6% (95%CI: 65.7-82.4) and 66.4% (95%CI: 58.2-75.8), respectively. Four independent adverse prognostic factors for OS were identified: age at diagnosis >2 years, time from diagnosis to HSCT >6 months, monocyte count at diagnosis >7.2x109/L, and the presence of additional genetic alterations. Based on these factors, we proposed a predictive classifier. Patients with three or more predictors (21% of the cohort) had a 5-year OS of 34.2%, whereas those with none (7%) had a 5-year OS of 100%. Our study demonstrates improved transplant outcomes compared to prior published data, which can be attributed to the synergistic impacts of a low TRM and a reduced yet still substantial relapse incidence. By integrating genetic information with clinical and hematological features, we have devised a predictive classifier. This classifier effectively identifies a subgroup of patients who are at a heightened risk of unfavorable post-transplant outcomes who would benefit novel therapeutic agents and post-transplant strategies.

The efficacy of combination treatment with elotuzumab and lenalidomide is dependent on crosstalk between natural killer cells, monocytes and myeloma cells.

Patients with refractory relapsed multiple myeloma respond to combination treatment with elotuzumab and lenalidomide. The mechanisms underlying this observation are not fully understood. Furthermore, biomarkers predictive of response have not been identified to date. To address these issues, we used a humanized myeloma mouse model and adoptive transfer of human natural killer (NK) cells to show that elotuzumab and lenalidomide treatment controlled myeloma growth, and this was mediated through CD16 on NK cells. In co-culture studies, we showed that peripheral blood mononuclear cells from a subset of patients with refractory relapsed multiple myeloma were effective killers of OPM2 myeloma cells when treated with elotuzumab and lenalidomide, and this was associated with significantly increased expression of CD54 on OPM2 cells. Furthermore, elotuzumab- and lenalidomide-induced OPM2 cell killing and increased OPM2 CD54 expression were dependent on both monocytes and NK cells, and these effects were not mediated by soluble factors alone. At the transcript level, elotuzumab and lenalidomide treatment significantly increased OPM2 myeloma cell expression of genes for trafficking and adhesion molecules, NK cell activation ligands and antigen presentation molecules. In conclusion, our findings suggest that multiple myeloma patients require elotuzumab- and lenalidomide-mediated upregulation of CD54 on autologous myeloma cells, in combination with NK cells and monocytes to mediate an effective anti-tumor response. Furthermore, our data suggest that increased myeloma cell CD54 expression levels could be a powerful predictive biomarker for response to elotuzumab and lenalidomide treatment.

Infections in children following chimeric antigen receptor T-cell therapy for B-cell acute lymphoblastic leukemia.

BACKGROUND: CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy is transforming care for pediatric patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). There are limited pediatric-specific data concerning the infection risks associated with CD19 CAR-T therapy and the adequacy of current antimicrobial prophylaxis guidelines for these patients. METHODS: We describe the antimicrobial prophylaxis used and the types of infectious occurring in the first 100 days following CAR-T therapy for relapsed or refractory B-cell ALL in children and adolescents (≤18 years) at our centre. RESULTS: Twenty-seven patients received their first CAR-T infusion (CTI) during the study period. Almost all patients (96%) had a comprehensive Infectious Diseases review prior to CTI, which informed a personalised prophylaxis or fever/sepsis plan in six (22%). Overall, six (22%) patients had one or more infections during the study period including five (19%, 0.9 per 100 days-at-risk) from days 0-30 and three (n = 20, 15%, 0.6 per 100 days-at-risk) from days 31-100. Bacterial blood stream infections were the most common type of infection encountered during both time periods, and one patient had probable pulmonary aspergillosis. There were no infection-related deaths. CONCLUSION: Our study contributes important information on the spectrum of infections encountered in pediatric patients with B-ALL post CAR-T therapy. Overall, the burden of infectious complications post CAR-T therapy in our cohort is lower than previously reported in the literature. Results suggest that our prophylaxis recommendations are effective in this population.

PVRIG is a novel natural killer cell immune checkpoint receptor in acute myeloid leukemia

This study explored the novel immune checkpoint poliovirus receptor-related immunoglobulin domain-containing (PVRIG) in acute myeloid leukemia (AML). We showed that AML patient blasts consistently expressed the PVRIG ligand (poliovirus receptor-related 2, PVRL2). Furthermore, PVRIG blockade significantly enhanced NK cell killing of PVRL2+, poliovirus receptor (PVR)lo AML cell lines, and significantly increased NK cell activation and degranulation in the context of patient primary AML blasts. However, in AML patient bone marrow, NK cell PVRIG expression levels were not increased. To understand how PVRIG blockade might potentially be exploited therapeutically, we investigated the biology of PVRIG and revealed that NK cell activation resulted in reduced PVRIG expression on the cell surface. This occurred whether NK cells were activated by tumour cell recognition, cytokines (IL-2 and IL-12) or activating receptor stimulation (CD16 and NKp46). PVRIG was present at higher levels in the cytoplasm than on the cell surface, particularly on CD56bright NK cells, which further increased cytoplasmic PVRIG levels following IL-2 and IL-12 activation. PVRIG was continually transported to the cell surface via the endoplasmic reticulum (ER) and Golgi in both unstimulated and activated NK cells. Taken together, our findings suggest that anti- PVRIG blocking antibody functions by binding to surface-bound PVRIG, which undergoes rapid turnover in both unstimulated and activated NK cells. We conclude that the PVRIGPVRL2 immune checkpoint axis can feasibly be targeted with PVRIG blocking antibody for NK-mediated immunotherapy of PVRL2+ AML.

The formin DIAPH1 (mDia1) regulates megakaryocyte proplatelet formation by remodeling the actin and microtubule cytoskeletons.

Megakaryocytes are highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rho-associated protein kinase (Rock)/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that 2 major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly, but also in the regulation of microtubule stability and dynamics during PPF.

TSTEM-like CAR-T cells exhibit improved persistence and tumor control compared with conventional CAR-T cells in preclinical models.

Patients who receive chimeric antigen receptor (CAR)-T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8+ memory T cell progenitors that can become either functional stem-like T (TSTEM) cells or dysfunctional T progenitor exhausted (TPEX) cells. To that end, we demonstrated that TSTEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate TSTEM-like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, TSTEM-like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4+ T cells during TSTEM-like CAR-T cell production. Adoptive transfer of TSTEM-like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of TSTEM-like CAR-T cells and an increased memory T cell pool. Last, TSTEM-like CAR-T cells and anti-programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8+CAR+ T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated TSTEM-like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.

A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer.

BACKGROUND: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. METHODS: We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. RESULTS: A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. CONCLUSIONS: Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

Advances in CAR T cell immunotherapy for paediatric brain tumours.

Brain tumours are the most common solid tumour in children and the leading cause of cancer related death in children. Current treatments include surgery, chemotherapy and radiotherapy. The need for aggressive treatment means many survivors are left with permanent severe disability, physical, intellectual and social. Recent progress in immunotherapy, including genetically engineered T cells with chimeric antigen receptors (CARs) for treating cancer, may provide new avenues to improved outcomes for patients with paediatric brain cancer. In this review we discuss advances in CAR T cell immunotherapy, the major CAR T cell targets that are in clinical and pre-clinical development with a focus on paediatric brain tumours, the paediatric brain tumour microenvironment and strategies used to improve CAR T cell therapy for paediatric tumours.

Enhancing the Potential of Immunotherapy in Paediatric Sarcomas: Breaking the Immunosuppressive Barrier with Receptor Tyrosine Kinase Inhibitors.

Despite aggressive surgery, chemotherapy, and radiotherapy, survival of children and adolescents and young adults (AYAs) with sarcoma has not improved significantly in the past four decades. Immune checkpoint inhibitors (ICIs) are an exciting type of immunotherapy that offer new opportunities for the treatment of paediatric and AYA sarcomas. However, to date, most children do not derive a benefit from this type of treatment as a monotherapy. The immunosuppressive tumour microenvironment is a major barrier limiting their efficacy. Combinations of ICIs, such as anti-PD-1 therapy, with targeted molecular therapies that have immunomodulatory properties may be the key to breaking through immunosuppressive barriers and improving patient outcomes. Preclinical studies have indicated that several receptor tyrosine kinase inhibitors (RTKi) can alter the tumour microenvironment and boost the efficacy of anti-PD-1 therapy. A number of these combinations have entered phase-1/2 clinical trials, mostly in adults, and in most instances have shown efficacy with manageable side-effects. In this review, we discuss the status of ICI therapy in paediatric and AYA sarcomas and the rationale for co-treatment with RTKis. We highlight new opportunities for the integration of ICI therapy with RTK inhibitors, to improve outcomes for children with sarcoma.

Chimeric Antigen Receptor T cell Therapy and the Immunosuppressive Tumor Microenvironment in Pediatric Sarcoma.

Sarcomas are a diverse group of bone and soft tissue tumors that account for over 10% of childhood cancers. Outcomes are particularly poor for children with refractory, relapsed, or metastatic disease. Chimeric antigen receptor T (CAR T) cells are an exciting form of adoptive cell therapy that potentially offers new hope for these children. In early trials, promising outcomes have been achieved in some pediatric patients with sarcoma. However, many children do not derive benefit despite significant expression of the targeted tumor antigen. The success of CAR T cell therapy in sarcomas and other solid tumors is limited by the immunosuppressive tumor microenvironment (TME). In this review, we provide an update of the CAR T cell therapies that are currently being tested in pediatric sarcoma clinical trials, including those targeting tumors that express HER2, NY-ESO, GD2, EGFR, GPC3, B7-H3, and MAGE-A4. We also outline promising new CAR T cells that are in pre-clinical development. Finally, we discuss strategies that are being used to overcome tumor-mediated immunosuppression in solid tumors; these strategies have the potential to improve clinical outcomes of CAR T cell therapy for children with sarcoma.

CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

CDK4/6 Inhibition Promotes Antitumor Immunity through the Induction of T-cell Memory.

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor-positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. SIGNIFICANCE: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer.This article is highlighted in the In This Issue feature, p. 2355.

Immune profiling of pediatric solid tumors.

Pediatric cancers, particularly high-risk solid tumors, urgently need effective and specific therapies. Their outlook has not appreciably improved in decades. Immunotherapies such as immune checkpoint inhibitors offer much promise, but most are only approved for use in adults. Though several hundred clinical trials have tested immune-based approaches in childhood cancers, few have been guided by biomarkers or clinical-grade assays developed to predict patient response and, ultimately, to help select those most likely to benefit. There is extensive evidence in adults to show that immune profiling has substantial predictive value, but few studies focus on childhood tumors, because of the relatively small disease population and restricted use of immune-based therapies. For instance, only one published study has retrospectively examined the immune profiles of pediatric brain tumors after immunotherapy. Furthermore, application and integration of advanced multiplex techniques has been extremely limited. Here, we review the current status of immune profiling of pediatric solid tumors, with emphasis on tumor types that represent enormous unmet clinical need, primarily in the context of immune checkpoint inhibitor therapy. Translating optimized and informative immune profiling into standard practice and access to personalized combination therapy will be critical if childhood cancers are to be treated effectively and affordably.

Lymphoblastic predominance of blastic phase in children with chronic myeloid leukaemia treated with imatinib: A report from the I-CML-Ped Study.

BACKGROUND: Chronic myeloid leukaemia (CML) is a rare disease in children. The frequency and outcome of children evolving to accelerated phase (AP) or blastic phase (BP) under treatment with imatinib is unknown. The aim of the current study is to assess the incidence of progression from CML in chronic phase with imatinib frontline in a paediatric setting and describe the management and outcome of these patients. PATIENTS AND METHODS: In the I-CML-Ped Study database (www.clinicaltrials.gov, #NCT01281735), 19 of 339 paediatric patients in chronic phase treated with imatinib in the frontline evolved to CML-AP or CML-BP. RESULTS: With a median follow-up of 38 months (range: 2-190 months), the cumulative incidence of progression at 1 and 3 years was 3% (confidence interval [CI] 95%: 1-5%) and 7% (CI 95%: 4-11%), respectively. We observed a large predominance of lymphoid-BP (70%) over myeloid-BP (30%) with imatinib in frontline therapy. Sixteen patients underwent haematopoietic stem cell transplantation, and eight were treated with a tyrosine kinase inhibitor after transplant. Only the transplanted patients are alive. The 5-year overall survival rate of children with CML-AP/BP is 44%, with no statistical difference between the lymphoid-BP and myeloid-BP outcome. CONCLUSION: Children evolving to AP or BP under treatment with imatinib have a very poor prognosis with an overall survival under 50%, much worse than children with advanced phase at diagnosis.